Project Summary/Abstract Antibacterial resistance is widespread for all major classes of antibiotics with many bacteria being resistant to multiple, and in some cases all, available antibiotics. To combat this resistance, novel antibiotics are needed. Natural products (NPs) from actinomycetes have traditionally been rich sources for antibiotics, but discovery of novel NPs from these strains via traditional means (i.e. screening crude culture supernatants for antibiotic activity) is rarely effective due to high rates of rediscovery. Whole genome sequencing of actinomycetes has revealed the presence of many unknown biosynthetic gene clusters suggesting the existence of many novel NPs. The Metcalf and Kelleher laboratories recently developed a method for untargeted NP discovery based upon genomic and mass spectrometric analyses (referred to as Natural Product Identification via Mass Spectrometry Based Analyses, NIMBLE). I propose to improve NIMBLE by incorporating computational structure predictions and molecular networking. I will use the improved NIMBLE to discover two novel antibiotic NPs and to elucidate the biosynthetic pathway for the more biologically active of the two. In Aim 1, twenty novel NPs identified via NIMBLE will be analyzed using computational structure prediction and molecular networking. This analysis will allow for identification of NPs with novel chemical scaffolds (not derivatives of known NPs!). Ten of the NPs with unique structures will be partially purified from their producing organisms and screened for activity against a panel of clinically relevant bacteria. Due to the high percentage of antibiotic NPs (~50% for actinomycete NPs), we expect that at least two antibiotic NPs will be identified. For the two most active NPs, the NIMBLE-predicted associations of the NPs with their biosynthetic gene clusters will be validated using in vivo genetic analysis. The compounds will then be isolated, and their structures will be elucidated. Finally, the purified NPs will be further analyzed for their antibacterial activity. In Aim 2, the biosynthetic pathway for the most potent NP identified in Aim 1 will be deciphered. An advantage of the NIMBLE method is that it allows simultaneous identification of novel NPs and their gene clusters. Having knowledge of the biosynthetic pathway will allow for future engineering of the strain for enhanced production of the NP and production of derivatives that could have improved activity or pharmacokinetic properties. The boundaries of the biosynthetic gene cluster and the functions of the genes within the cluster will be determined via bioinformatics analyses, genetic validation, and if needed, in vitro analysis of the enzymes encoded in the cluster. The order of the pathway will be determined via cross-feeding studies. Overall, this proposal will allow for identification of 2 novel antibiotic natural products and elucidation of the biosynthetic pathway for one of them. More importantly, it will provide a general platform for discovering many more bioactive natural products.